Project Summary: Drugs specifically developed against class A priority bacterial pathogens do not exist. Infections of anthrax, plague, and tularemia are currently treated with existing antibiotics such as ciprofloxacin and doxycycline. However, antibiotic-resistant strains of the bacterial bioterrorism agents are known, rendering current drugs ineffective, and furthermore, existing drugs are not optimized to treat the above agents of interest. The long term goal of this proposal is to develop two novel antibacterial drug classes, each of which has been optimized to be efficacious against disease caused by any of the three class A pathogens, B. anthracis, Y. pestis, or F. tularensis. That is, the treatment of choice against any of these pathogens could be the same drug, thus enabling immediate and efficacious treatment in the absence of a definitive diagnosis. This could mean the difference between life and death in a bioterrorism attack, since symptoms due to aerosol exposure to these agents would be indistinguishable. The enzymes nicotinate mononucleotide adenylyl- transferase (NAMNAT) and NAD+ synthetase (NADS), which catalyze the last 2 steps in NAD* biosynthesis, have been shown to be absolutely essential to the survival of every bacterium studied to date. Drugs developed against either could be used alone or together for an effective combination therapy that may be less susceptible to resistance strains. We developed the first reported small molecule inhibitors of NADS with antibacterial activity and selectivity for the bacterial versus human enzyme. Bacterial enzymes for each target (three per target;six in all) will be used to optimize lead compounds that are simultaneously effective against all three organisms. Within the funding period of this U01, inhibitors of NAMNAT and NADS will be developed through a reiterative cycle of molecular modeling and virtual screening against enzyme structures, medicinal chemistry/compound library development/structure-activity analysis, compound screening, and initial preclinical toxicology, pharmacokinetic, and animal efficacy against three Category A pathogens, B. anthracis, Y. pestis, and F. tularensis. At the same time, the human homolog will be an integral part of the design strategy so that inhibitors can be simultaneously designed for minimal human toxicity. In fact, selective inhibitors of bacterial NADS and NAMNAT are known. The goal of this U01 program is to produce a collection of advanced lead compounds that are safe, orally bioavailable, and efficacious in an established murine model. Relevance: The research conducted will lead to new drugs for the treatment of anthrax, plague, and tularemia. These diseases are caused by three of the highest risk bacterial bioterrorism agents, B. anthracis, Y. pestis, and F. tularensis.